To cause a change in a state of molecular aggregation, generally the concentration of the surfactant or temperature is changed. In an equilibrium system such as a micelle, the state can be rapidly changed by adding a different type of surfactant.
On the other hand, in dispersed systems such as bilayer membrane vesicles, of which liposomes are a representative example, lipids that constitute the dispersed system are in a relatively stable state and thus transfer very slowly. To induce fusion or fission of these aggregates, generally a change in a physical state of an interface is occurred. Such a change is highly dependent on the lipids to be used, the reaction conditions and the like and often there are limitations.
For example, liposomes composed of phosphatidylserine can be made to undergo a phase transition such as membrane fusion by adding Ca2+ (Duzgunes et al., Biochemistry, (1987) vol. 26, pp. 8435-8442). It is believed that this is because Ca2+ causes charge neutralization, crosslink between lipids, and dehydration so that the membrane becomes unstable. However, this method cannot be employed for liposomes composed of only neutral phospholipids. It has also been reported that membrane fusion occurs upon adding high concentration of polyethylene glycol to liposomes composed of phosphatidylcholine (Lentz et al., Biochemistry, (1992) vol. 31, pp. 2643-2653, and Yang et al., Biophysical Journal, (1997) vol. 73, pp. 277-282). This fusion is caused by destabilization of the membrane due to loss of the free water in the membrane. A membrane fusion method by utilizing viruses also has been proposed (Blumenthal et al., Chemistry and Physics of Lipids, (2002) vol. 116, pp. 39-55). This method requires receptors against virus outside the membrane. Other reported methods include a method of inducing membrane fusion via physical stimulus caused by an electrical pulse (Sugar et al., Biophysical Chemistry, (1987) vol. 26, p. 321) and a method of membrane fusion by irradiating UV light on adhered liposomes (Kuhn et al., Langmuir, (2003) vol. 19, pp. 8206-8210). There is also a method of adding protein or peptide to cause a change in the higher-order structure due to pH-dependant protonization and thereby inducing membrane fusion (Kim et al., Biochemistry, (1986) vol. 25, pp. 7867-7874).
Every one of these various fusion methods is based on a change in the physical state of the lipids forming the molecular aggregate, and requires aggregation at a previous stage of fusion. In other words, when lipids alone are dispersed, the lipids themselves that constitute the molecular aggregate are in a completely inactivated state.
On the other hand, there has been reported phase transitions of the membrane, such as fusion and fission, based on chemical reaction (Takakura et al., Chemistry Letters, (2002) pp. 404-405, and Toyota et al., Chemistry Letters, (2004) vol. 33, pp. 1442-1443). Specifically, imine formation and subsequent hydrolysis thereof due to dehydrating condensation in the bilayer membrane of the vesicle cause to morphological change in the vesicle and thus leads to membrane fusion and fission. For example, when a dispersion of micelle of amphipathic lipids having a hydrophilic reactive group (amino group) is added to a dispersion of vesicles composed of amphipathic lipids having a hydrophobic reactive group (aldehyde group), an imine is produced through reversible dehydrating condensation between the reactive groups in the membrane bilayer, so that the vesicles become larger (Takakura et al., ibid.). Further, depending on the abundance ratio of the lipids having these reactive groups and the amphipathic lipids produced by dehydrating condensation, a reversible morphological change in the vesicles has been observed (Toyota et al., ibid.). However, these methods do not allow the state of the bilayer membrane of the vesicles to be controlled.
There also are methods of causing fusion by changing the lipid structure through biological means using enzymes. Specifically, these methods involve hydrolyzing phosphatidylcholine or phosphatidylethanolamine with phospholipase C (Nieva, J.-L., et al., Biochemistry, (1989) vol. 28, pp. 7364-7367) or hydrolyzing sphingomyelin with sphingomyelinase (Kazuo Ooki, Seibutsu Butsuri, (2004) vol. 44, pp. 161-165) to remove the phosphate group of the phospholipid constituting the bilayer membrane of the vesicle, thereby producing diacylglycerol or ceramide, respectively. In either case, the morphology of the molecule changes from an inverse cone shape to a cylinder in which the molecular area of the polar head portion is small (the critical load parameter changes), so that the curvature alters to cause fusion.
Biological research has shown that the activity of enzymes that acylate single-strand phospholipids to convert them to double-strand phospholipids is increased when membrane fusion or fission is caused (Schmidt, A., et al., Nature, (1999) vol. 401, pp. 133-141). That is to say, it has been shown that in nerve terminal synapses, lysophosphatidic acid (LPA) acyltransferase is essential for reconstituting synapse vesicles. This enzyme transfers an acyl group to LPA, which is monoacylglycerol bonded with a phosphate (single-strand phospholipid), to convert it to a double-stranded phospholipids. Since this reaction occurs when the membrane undergoes a change, it is suggested that the change in curvature in the membrane caused by such enzymatic chemical reactions is important.